The effect of Lactobacillus buchneri and L. plantarum, applied at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages

The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (LP) and yeasts at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages was studied under laboratory conditions. Treatments comprised LB, LP, yeasts, LB + yeasts, LP + yeasts, LB + LP and B-589 (a lactic acid bacterial strain isolated from wheat silage in Israel) alone. The treatments were also applied to sterilized aqueous extracts of wheat which were incubated at 30°C for 10 days. The pH of all treatments was below 4.0 already on day 4 of the experiment. Silages treated with LB had higher acetic acid concentrations than those treated with LP: 32–34 vs 16–18, and 28–34 vs 4–7 g kg⁻¹ in the experiments with wheat and sorghum, respectively. Similar results were obtained in wheat extracts. In the aqueous phase, marked differences in pH decrease were noticed among the treatments: 4.4 in LB, 6.0 in the yeast, and 3.7 in LP and B-589 (from day 3 and onwards). In both crops LB resulted in aerobically stable silages when applied alone or with LP and yeasts, whereas LP resulted in unstable silages upon aerobic exposure; the stability of the LB-treated silages is attributed to the higher acetic acid concentrations. The isolated strain (B-589) did not exhibit any advantage with regard to aerobic stability. Received 26 April 1999/ Accepted in revised form 05 July 1999     

The effects of temperature on the aerobic stability of wheat and corn silages

The aim of this work was to study the effects of temperature on the aerobic stability of wheat and corn silages. Three silage samples from each crop were taken from the faces of six different commercial bunker silos immediately after unloading them. The samples were exposed to air for 3 or 6 days at 10, 20, 30 or 40°C. The most intensive deterioration occurred at 30°C. Samples incubated at 30°C had the highest yeast counts, most prolific CO₂ production and greatest increases in pH. Silage samples exposed to 10 or 40°C remained stable. The duration of exposure had a significant effect on aerobic stability, especially at 30°C. Temperature has a significant effect on silage aerobic stability. In a warm climate, special care should be taken during unloading of silage in order to prevent intensive aerobic deterioration. Journal of Industrial Microbiology & Biotechnology (2002) 28, 261–263 DOI: 10.1038/sj/jim/7000237 Received 12 June 2001/ Accepted in revised form 02 November 2001     

The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages

AIMS: To determine the effect of Propionibacterium acidipropionici, alone or in combination with Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages. METHODS AND RESULTS: The inoculants were applied at 1.0 x 10(6) CFU g(-1). Silages with no additives served as control. Fresh forages were sampled prior to ensiling. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, the silages were subjected to an aerobic stability test. The P. acidipropionici-inoculated silages had significantly higher levels of acetic and propionic acid than the L. plantarum or P. acidipropionici + L. plantarum-inoculated silages (P < 0.05). Therefore, yeast activity was impaired in the P. acidipropionici-inoculated silages. As a result, P. acidipropionici decreased CO(2) production and improved aerobic stability of wheat, sorghum and maize silages. However, the combination of P. acidipropionici + L. plantarum did not improve aerobic stability of the silages. CONCLUSIONS: The P. acidipropionici was very effective in protecting the wheat, sorghum and maize silages exposed to air under laboratory conditions, probably because the acidic environment under ensiling conditions is favourable for this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of P. acidipropionici, as a silage inoculant can improve the aerobic stability of silages by inhibition of yeast activity.     

The effect of applying lactic acid bacteria at ensiling on the aerobic stability of silages

The effect of applying commercial lactic acid bacteria inoculants at ensiling on the aerobic stability of silages was studied under laboratory conditions. The silages used were wheat, hedysarum, corn and various sorghum cultivars at various stages of maturity. Three inoculants were used, two containing Lactobacillus plantarum, Enterococcus faecium and Pediococcus acidilactici (H/M F, Medipharm, USA and Sil-All, Alltech, UK) and one containing Ent. faecium (Lacticil, M74, Medipharm, Sweden). The inoculants were applied at 0.5 times 10⁶ cfu g^-1. Silages with no additives served as controls. After treatment, the chopped forages were ensiled in 1.5 1 anaerobic jars; there were six jars per treatment. After ensiling for 45 d, the silages were tested for aerobic stability in a test in which CO₂ production was measured along with chemical and microbiological parameters.
The inoculated silages that spoiled upon aerobic exposure faster than the controls were those of wheat and of the sorghum cultivar FS5 at the milk stage of maturity. This was evident from intensive CO₂ production and development of yeasts and moulds. Regression analysis indicated that aerobic deterioration of inoculated silages was associated with high levels of residual water-soluble carbohydrates and lactic acid and lack of volatile fatty acids. Aerobic spoilage of inoculated silages was attributed mainly to yeast activity.     

The effects of Propionibacterium acidipropionici and Lactobacillus plantarum, applied at ensiling, on the fermentation and aerobic stability of low dry matter corn and sorghum silages

The aim of this work was to study the effects of applying a strain of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability characteristics of low dry matter (DM) corn (Zea mays L.) and sorghum (Sorghum bicolor L.) silages. Corn at the dent stage and sorghum at the flowering stage were harvested. Treatments comprised control (no additives), P. acidipropionici, L. plantarum and a combination of P. acidipropionici and L. plantarum. Fresh forages were sampled prior to ensiling. Bacterial inoculants were applied to the fresh forage at 1.0×10⁶ colony-forming units per gram. After treatment, the chopped fresh materials were ensiled in 1.5-l anaerobic glass jars equipped with a lid that enabled gas release only. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, 60 days, the silages were subjected to an aerobic stability test. The L. plantarum inoculated silages had significantly higher levels of lactic acid than the controls, P. acidipropionici and combination of P. acidipropionici and L. plantarum inoculated silages (P<0.05). The P. acidipropionici did not increase propionic and acetic acid levels of the silages. After the aerobic exposure test, the L. plantarum and combination of P. acidipropionici and L. plantarum had produced more CO₂ than the controls and the silages inoculated with P. acidipropionici (P<0.05). All silages had high levels of CO₂ and high numbers of yeasts and molds in the experiment. Therefore, all silages were deteriorated under aerobic conditions. The P. acidipropionici and combination of P. acidipropionici and L. plantarum were not able to improve the aerobic stability of fast-fermenting silages, because they could not work well in this acidic environment. The results showed that P. acidipropionici and combination of P. acidipropionici and L. plantarum did not improve the aerobic stability of low DM corn and sorghum silages, which are prone to aerobic deterioration.     

The effect of Lactobacillus buchneri on the fermentation, aerobic stability and ruminal degradability of maize silage

AIMS: To evaluate the effect of Lactobacillus buchneri, heterofermentative lactic acid bacteria (LAB), on the fermentation, aerobic stability and ruminal degradability of whole-crop maize silages under laboratory conditions. Two homofermentative LAB were tested for the purpose of comparison. METHODS AND RESULTS: Maize was harvested at early dent [290 g kg(-1) dry matter (DM)] and one-half milk line (355 g kg(-1) DM) stages. Both homofermentative LAB were applied at 1 x 10(5) CFU g(-1) of fresh forage. Lactobacillus buchneri was applied at 1 x 10(5), 5 x 10(5) and 1 x 10(6) CFU g(-1) of fresh forage. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-l anaerobic jars. Three jars per treatment were sampled on day 60. After 60 days of storage, silages were subjected to an aerobic stability test lasting for 5 days, in which CO(2) production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. Both homofermentative LAB increased the concentration of lactic acid and the numbers of yeasts, and decreased the concentration of acetic acid and impaired the aerobic stability of silages. In contrast, applying L. buchneri decreased the concentration of lactic acid and increased the concentration of acetic acid of the silages. Under aerobic conditions, silages treated with 5 x 10(5) and 1 x 10(6) CFU g(-1) of L. buchneri, had lower pH, CO(2) production and the numbers of yeasts than the silages treated with 1 x 10(5) CFU g(-1) of L. buchneri (P < 0.05). However, all doses of L. buchneri and both homofermentative LAB did not affect in situ rumen DM, organic matter and neutral detergent fibre degradability of the silages. CONCLUSIONS: Lactobacillus buchneri was very effective in protecting maize silages exposed to air under laboratory conditions. All doses of L. buchneri, especially 5 x 10(5) CFU g(-1) or more, markedly decreased the numbers of yeasts and improved the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of L. buchneri, as a silage inoculant, can improve the aerobic stability of maize silages by inhibition of yeast activity.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.     

Accumulation of 1,2-propanediol and enhancement of aerobic stability in whole crop maize silage inoculated with Lactobacillus buchneri

Aims: To assess the effects of inoculation of Lactobacillus buchneri on the ensiling properties and aerobic stability of maize silage. Methods and Results: Chopped whole crop maize was ensiled in 0.5 litre airtight polyethylene bottles (0.4 kg per bottle) and in double-layered, thin polyethylene bags (15 kg per bag), with or without inoculation of Lact. buchneri. The silos were stored for two to four months and the chemical composition, microbial numbers and aerobic stability were determined. Inoculation lowered lactic acid and yeasts, and increased acetic acid and pH value, resulting in improved aerobic stability of the silages. Inoculated silages produced 1,2-propanediol, the content of which increased as ensiling was prolonged, and nearly 50 g kg-1 dry matter had accumulated after four months of storage. The effects of inoculation, however, were much less pronounced in silages prepared in bags. Mannitol was found in all silages; the production was lowered by Lact. buchneri treatment and appeared to be unrelated to the accumulation of 1,2-propanediol. Conclusions: Inoculation of Lact. buchneri occasionally causes accumulation of 1,2-propanediol in silages without further degradation into propionic acid and 1-propanol. Significance and Impact of the Study: Substantial amounts of 1,2-propanediol could be consumed by ruminants when fed on silages inoculated with Lact. buchneri. In addition to increasing acetic acid, attention needs to be paid to 1,2-propanediol because the two fermentation products might affect the intake and utilization of silage-based diets.     

Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants

Aims:  The effect of the inoculation of maize and sorghum silages with Lactobacillus plantarum (LP) and Lactobacillus buchneri (LB) on the clostridia spore formation during aerobic deterioration has been studied.
Methods and results:  The crops were ensiled in 30 l jars, without a lactic acid bacteria inoculant (C), and with an LP or LB inocula (theoretical rate of 1 × 10⁶). After 90 days of conservation, the silages were analysed for the chemical and microbiological characteristics and subjected to an aerobic stability test, during which pH, temperature, nitrate, yeast, mould and clostridia spores were measured. Compared to the C and LP silages, yeasts were reduced in the LB silages, resulting in an increased aerobic stability. Clostridia spores, determined by most probable number (MPN) procedure, increased to 6 log₁₀ MPN g⁻¹ in the C and LP maize silages, whereas they reached 3 log₁₀ MPN g⁻¹ in C and LP sorghum silages.
Conclusions:  Clostridia spore count only slightly increased in the LB maize silages after 342 h (2·59 log₁₀ MPN g⁻¹), whereas it did not show any increase in the LB sorghum silages for the whole period of air exposure.
Significance and impact of the study:  The data indicated that clostridia spore outgrowth can take place during silo feedout in aerobic-deteriorated silages and that LB inoculation reduces the risk of clostridia outgrowth after silage opening by increasing the aerobic stability.     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

The effect of an inoculant and enzymes on fermentation and nutritive value of sorghum straw silages

The objectives of this study were to determine the effect of inoculant, enzymes and inoculant-enzymes mixture on fermentation quality, nutritive value, and microbial changes of sorghum straw silage. Sorghum straws were collected and treated with distilled water (control), inoculant, enzymes and inoculant+enzymes prior to ensiling. Three bag silos for each silage (denoted C, I, E and I+E, respectively) were opened after 3, 7, 11, 15, 30 and 60 days for chemical and microbial analyses. For all the silages, there was a rapid decline in pH during the first 3 days of ensiling. Relative to silage C, all the treatment (I, E and I+E) had higher (P<0.05) lactic acid concentration at all ensiling periods. Population of LAB during all ensiling time was numerically greater for treated than control silages. Separate addition of two additives, especially for enzymes, can effectively (P<0.05) decrease aNDF and ADF concentration. Treatments with enzymes (E, I+E) can also improve significantly silage IVDMD and IVNDFD concentration. These results indicated that the addition of additives can improve the sorghum straw silage fermentation quality at different extent.     

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling

The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.